Direct reprogramming of human fibroblasts into insulin-producing cells using transcription factors

Direct lineage reprogramming of one somatic cell into another without transitioning through a progenitor stage has emerged as a strategy to generate clinically relevant cell types. One cell type of interest is the pancreatic insulin-producing β cell whose loss and/or dysfunction leads to diabetes. To date it has been possible to create β-like cells from related endodermal cell types by forcing the expression of developmental transcription factors, but not from more distant cell lineages like fibroblasts. In light of the therapeutic benefits of choosing an accessible cell type as the cell of origin, in this study we set out to analyze the feasibility of transforming human skin fibroblasts into β-like cells. We describe how the timed-introduction of five developmental transcription factors (Neurog3, Pdx1, MafA, Pax4, and Nkx2-2) promotes conversion of fibroblasts toward a β-cell fate. Reprogrammed cells exhibit β-cell features including β-cell gene expression and glucose-responsive intracellular calcium mobilization. Moreover, reprogrammed cells display glucose-induced insulin secretion in vitro and in vivo. This work provides proof-of-concept of the capacity to make insulin-producing cells from human fibroblasts via transcription factor-mediated direct reprogramming.


Supplementary Figure 1. Effects of culture media formulation on INS gene induction by Ad-NPM in human fibroblasts.
Human fibroblasts (HFF1) were infected with Ad-NPM. After virus removal, cells were cultured in normal fibroblast growth media (DMEM-F12, 4500mg/L glucose and 10% (v/v) Fetal Bovine Serum or FBS) or changed to the culture media and FBS concentrations indicated in the X-axis. Glucose concentrations in DMEM low glucose, RPMI-1640 and CMRL-1066 were 1000mg/L, 2000 mg/L and 1000 mg/L respectively. Seven days later, cells were harvested and RNA extracted to determine INS mRNA by qPCR. (a) INS mRNA levels expressed relative to TBP. Values are mean ± SEM of 3-7 independent experiments. (b) Representative real time PCR amplification plots for INS and TBP in parental fibroblasts and in fibroblasts seven days after infection with Ad-NPM. ****, P<0.0001 relative to parental fibroblasts (C) using one-way ANOVA followed by Dunnett's multiple comparison test.

Supplementary Figure 2. Induction of the INS gene by different transcription factor combinations in human fibroblasts.
Human fibroblasts (HFF1) were infected with recombinant adenoviruses encoding the transcription factors indicated in the X-axis. Except for NPM, which refers to the polycistronic adenovirus encoding Neurog3, Pdx1 and Mafa, all other adenoviruses encoded individual transcription factors. Adenoviruses were added either sequentially (order of addition is shown left to right) or simultaneously (shown in parenthesis). The interval between subsequent infections was two days. (a) qPCR for INS seven days after initial infection. (b) qPCR of INS ten days after initial infection. Expression levels are calculated relative to TBP. Data are mean ± SEM for n=2-5 independent experiments. **,P<0.01; ***,P<0.001 relative to parental fibroblasts (C) using one-way ANOVA followed by Dunnett's multiple comparison test.

Supplementary Figure 3. Staining for islet hormones in 5TF cell spheroids.
Representative confocal images of 5TF cell spheroids collected at day 11 of the 5TF-3D reprogramming protocol and immunostained with the indicated antibodies. Insulin is shown in yellow, glucagon in red and somatostatin in light blue. In the merge image, nuclei are shown in white (marked with Hoechst). Spheroids consisting of parental fibroblasts and fixed human pancreas are shown as negative and positive control, respectively. Scale bars, 50 µm.

Supplementary Figure 4. Validation of the 5TF-3D protocol in a second HFF preparation.
Human foreskin fibroblasts (HFF2) were subjected to transcription factor-based reprogramming toward insulin-producing cells. (a) Bright field and Cherry immunofluorescence images from control (parental) fibroblasts and from fibroblasts three days after infection with Ad-NPM. Scale bar, 100 µm. (b) qPCR for INS in HFF2 ten days after infection with Ad-NPM alone and cultured under 2D conditions in DMEM/10%FBS (DM) or RPMI-1640/6% FBS media (RP) (n=6), or after the 5TF reprogramming protocol and cultured in RP media under 2D or 3D conditions (n=4). Expression levels were calculated relative to TBP. Note that changes in INS gene expression in the different reprogramming conditions follow a similar trend as observed with HFF1 fibroblasts. (c) qPCR of the indicated genes in HFF2-derived 5TF cell spheroids (n=6). Transcript levels are expressed relative to spheroids maintained in 2D culture throughout the 10-day protocol (given the value of 1). (d) Representative immunofluorescence image showing insulin (using antibody ab2 against C-peptide) in green and nuclei in blue (marked with Hoechst). Scale bar, 100 µm. (e) Glucose stimulation Index (ratio between insulin secreted at 20mM glucose vs. 2mM glucose) of 5TF cells spheroids (n=7, from 3 reprogramming experiments). Data are mean ± SEM. *, P< 0.05; **, P< 0.01; ***, P< 0.001 between indicated conditions in (b) using unpaired t-test and relative to 2D in (c) using one-sample test.

Supplementary Figure 5. Identification of insulin-positive cells in 10-day eye grafts.
Eye grafts were harvested ten days following transplantation of 5TF cell spheroids into the anterior chamber of the eye of normoglycemic NSG mice. After fixation, grafts were immunostained with the indicated antibodies.

Supplementary Figure 9. Improvement of adenoviral infection efficiency in human fibroblasts by including DNA transfection reagents.
Human fibroblasts (HFF1) were infected with a polycistronic recombinant adenovirus encoding Neurog3, Pdx1, Mafa and a Cherry reporter protein (Ad-NPM), in the presence or absence of the transfection reagents Superfect and jetPEI. (a) Bright field and Cherry immunofluorescence images from control (parental) fibroblasts and HFF1 fibroblasts three days after infection with Ad-NPM alone or Ad-NPM + Superfect. (b) qPCR of the transgenes three days after infection with Ad-NPM in the presence or absence of the indicated transfection reagent. Data are presented as the mean ± SEM for n= 2 for NPM alone and n=3-4 for the other conditions. Scale bar, 100 µm.